The proposed research involves both genetic and biochemical studies on genetic regulatory mechanisms in the qa system of the eucaryote Neurospora crassa. The qa system comprises a cluster of one regulatory gene (qa-1) plus three structural genes which encode three inducible enzymes catalyzing sequential steps in the catabolism of quinic acid. The product of the regulatory gene is a protein which appears to act in a positive fashion as an activator in combination with the inducer, quinic acid, to initiate coordinate synthesis of the qa enzymes. Major efforts will be concentrated in two areas of study: 1. the organization and functioning of the qa gene cluster in N. crassa and 2. its expression when cloned on an Escherichia coli plasmid. Combined genetic and biochemical studies will be continued to establish the orientation and direction of transcription of the three qa genes which encode enzymes. Efforts will be continued to identify and map the positions of initiators. Experiments will be continued to examine regulation in vitro by utilizing cell-free transcriptional and translation systems. Attempts will be made to determine at least a substantial fraction of the amino acid sequence of catabolic dehydroquinase (the product of the qa-2 plus gene) which is synthesized by a recombinant E. coli plasmid for comparison with the available N. crassa seqence. Additional experiments will be undertaken to determine if the qa-3 plus and qa-4 plus gene products can be experessed in E. coli. Experiments to transform N. crassa utilizing available hybrid E. coli plasmids are also planned, since these may help interpret problems surrounding the expression of eucaryotic DNA in E. coli. Nucleotide seqencing of the cloned Neurospora qa DNA will be initiated in order to identify regulatory sequences.